is widely used in biotechnology to produce recombinant proteins, food ingredients and diverse natural products. However, unstable expression of plasmids, difficult and time-consuming integration of single and low-copy-number plasmids hampers the construction of efficient production pathways and application to industrial production. Here, by exploiting sequence diversity in the long terminal repeats (LTRs) of retrotransposons and ribosomal DNA (rDNA) sequences, a set of vectors and methods that can recycle multiple and high-copy-number plasmids was developed that can achieve stable integration of long-pathway genes in . By combining these sequences, amino acids and antibiotic tags with the Cre-LoxP system, a series of multi-copy site integration recyclable vectors were constructed and assessed using the green fluorescent protein (HrGFP) reporter system. Furthermore, by combining the consensus sequence with the vector backbone of a rapidly degrading selective marker and a weak promoter, multiple integrated high-copy-number vectors were obtained and high levels of stable HrGFP expression were achieved. To validate the universality of the tools, simple integration of essential biosynthesis modules was explored, and 7.3 g/L of L-ergothioneine and 8.3 g/L of (2)naringenin were achieved in a 5 L fermenter, the highest titres reported to date for . These novel multi-copy genome integration strategies provide convenient and effective tools for further metabolic engineering of .

中文翻译：

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YaliCMulti 和 YaliHMulti：用于工程解脂耶氏酵母的稳定、高效的多拷贝集成工具

广泛应用于生物技术生产重组蛋白、食品成分和多种天然产品。然而，质粒表达不稳定、单拷贝数和低拷贝数质粒的整合困难且耗时，阻碍了高效生产途径的构建和工业化应用。这里，通过利用反转录转座子和核糖体DNA（rDNA）序列的长末端重复（LTR）中的序列多样性，开发了一套可以回收多个高拷贝数质粒的载体和方法，可以实现长末端重复序列的稳定整合。 - 中的途径基因。通过将这些序列、氨基酸和抗生素标签与Cre-LoxP系统相结合，构建了一系列多拷贝位点整合可回收载体，并使用绿色荧光蛋白（HrGFP）报告系统进行评估。此外，通过将共有序列与快速降解选择标记和弱启动子的载体骨架相结合，获得了多个整合的高拷贝数载体，并实现了高水平的稳定HrGFP表达。为了验证工具的通用性，探索了基本生物合成模块的简单集成，并在 5 L 发酵罐中获得了 7.3 g/L 的 L-麦角硫因和 8.3 g/L (2)柚皮素，这是迄今为止报道的最高滴度为了 。这些新颖的多拷贝基因组整合策略为进一步的代谢工程提供了方便有效的工具。